Levels of CXCL 10/IP-10 forms and soluble CD26/DPPIV activity as early predictive biomarkers for HIV/SIV associated mucosal inflammation and progression towards AIDS

ABSTRACT

The invention provides methods for the identification of patients capable of controlling HIV progression, as well as to the identification of an antagonist form of IP-10 associated to HIV progression control and the uses thereof for improving the immunological response of HIV patients.

FIELD OF THE INVENTION

The invention relates to the field of immunology and, in particular, tomethods for the identification of patients capable of controlling HIVprogression, as well as to the identification of an antagonist form ofIP-10 associated to HIV progression control and the uses thereof forimproving the immunological response of HIV patients.

BACKGROUND OF THE INVENTION

HIV disease is a continuum of progressive damage to the immune systemfrom the time of infection to the manifestation of severe immunologicdamage by opportunistic infections, neoplasms, wasting, or low CD4lymphocyte count that define AIDS. In the absence of antiretroviraltherapy, this results in an important immunologic impairment with theconsequent appearance of opportunistic infections and, lastly, death.The time it takes to traverse this spectrum varies greatly, ranging from1 year or less in some persons to a still unknown upper limit in othersthat has reached nearly 20 years in a few individuals.

Significant advances in antiretroviral therapy of HIV infection havebeen made since the introduction of zidovudine (AZT) in 1987. Intensiveresearch on HIV led to the development of highly active antiretroviraltherapies (HAART). HAART is defined as treatment with at least threeactive anti-retroviral medications, commonly reverse transcriptaseinhibitors and protease inhibitors. These therapies have been extremelysuccessful in controlling the spread of the disease. Indeed, with theadvent of HAART, HIV infection is now manageable as a chronic disease inpatients who have access to medication and who achieve durablevirological suppression.

However, residual chronic inflammation persists in HAART-treatedpatients and is associated with increased risk of cardiovasculardiseases and mortality. Both AIDS and non-AIDS mortality are attributedto chronic inflammation in HIV-infected individuals. No therapeuticstrategy exists to reduce this inflammation efficiently. This is in partexplained by the fact that the inflammatory pathways involved are notwell identified. Chronic immune activation is considered as the majordriving force for CD4⁺ T cell depletion and progression towards AIDS inHIV-infected individuals. HIV-triggered chronic inflammation remainshigher even in patients who control viral load (patients under efficientanti-retroviral treatment and HIV controllers in comparison to healthydonors. Current antiretroviral treatments are highly efficient, but failto abolish residual chronic immune activation.

There is thus an urgent need to understand the factors, which drive thisinflammation and to define good surrogate markers for this inflammation.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is related to the discovery that levels ofcertain biomarkers, including the short form of IP-10 (sIP-10), in theplasma of HIV patients are negative predictors of those patients whowill progress towards AIDS.

Accordingly, in one aspect of the invention, a method is provided forassessing the likelihood of a patient to develop AIDS. Generally, themethod includes at least the following steps: (1) assaying a biologicalsample from a patient identified as infected with HIV to determine thelevel of sIP-10 in the biological sample; and (2) assessing thelikelihood that the patient will develop AIDS based on the level ofsIP-10.

In another aspect of the invention, a method is provided for prognosingof a patient identified as suffering from HIV infection. Generally, themethod includes at least the following steps: (1) determining the levelof sIP-10 in a biological sample taken from the patient; and (2)determining the prognosis of the patient based on the level of sIP-10.

In another aspect of the invention, a method is provided for treatingHIV infection. Generally, the method includes at least the followingsteps: (1) obtaining a biological sample from a patient identified ashaving HIV infection; (2) determining the level of sIP-10 in thebiological sample; and (3) selecting a drug therapy based on the levelof sIP-10 in the sample.

More precisely, the present disclosure is related to the discovery thatthe relative levels of sIP-10 are negative predictors of those patientswho will progress towards AIDS.

Accordingly, in one aspect of the invention, a method is provided forassessing the likelihood of a patient to develop AIDS. Generally, themethod includes at least the following steps: (1) assaying a biologicalsample from a patient identified as infected with HIV to determine thelevel of sIP-10 in the biological sample; (2) determining the ratio ofsIP-10 to total IP-10, and (3) assessing the likelihood that the patientwill develop AIDS based on the ratio of sIP-10 to total IP-10.

In another aspect of the invention, a method is provided for prognosingof a patient identified as suffering from HIV infection. Generally, themethod includes at least the following steps: (1) determining the levelof sIP-10 in a biological sample taken from the patient; (2) determiningthe ratio of sIP-10 to total IP-10 in said biological sample, and (3)determining the prognosis of the patient based on the ratio of sIP-10 tototal IP-10.

In another aspect of the invention, a method is provided for treatingHIV infection. Generally, the method includes at least the followingsteps: (1) obtaining a biological sample from a patient identified ashaving HIV infection; (2) determining the level of sIP-10 in thebiological sample; (3) determining the ratio of sIP-10 to total IP-10 inthe biological sample, and (4) selecting a drug therapy based on theratio of sIP-10 to total IP-10 in the sample.

The present disclosure is also related to the discovery that theenzymatic activities of certain biomarkers, including thedipeptidylpeptidase IV enzyme (DPPIV), in the plasma of HCV patients arenegative predictors of those patients who will progress towards AIDS.

Accordingly, in one aspect of the invention, a method is provided forassessing the likelihood of a patient to develop AIDS. Generally, themethod includes at least the following steps: (1) assaying a biologicalsample from a patient identified as infected with HIV to determine theactivity of DPPIV in the biological sample; and (2) assessing thelikelihood that the patient will develop AIDS based on the activity ofDPPIV.

In another aspect of the invention, a method is provided for prognosingof a patient identified as suffering from HIV infection. Generally, themethod includes at least the following steps: (1) determining theactivity of DPPIV in a biological sample taken from the patient; and (2)determining the prognosis of the patient based on the activity of DPPIV.

In another aspect of the invention, a method is provided for treatingHIV infection. Generally, the method includes at least the followingsteps: (1) obtaining a biological sample from a patient identified ashaving HIV infection; (2) determining the activity of DPPIV in thebiological sample; and (3) selecting a drug therapy based on theactivity of DPPIV in the sample.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. The practiceof the invention employs, unless other otherwise indicated, conventionaltechniques or protein chemistry, molecular virology, microbiology,recombinant DNA technology, and pharmacology, which are within the skillof the art. Such techniques are explained fully in the literature (seee.g., Ausubel et al., Short Protocols in Molecular Biology, CurrentProtocols; 5th Ed., 2002; Remington's Pharmaceutical Sciences, 17th ed.,Mack Publishing Co., Easton, Pa., 1985; and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rdEd., 2001). The nomenclatures used in connection with, and thelaboratory procedures and techniques of, molecular and cellular biology,protein biochemistry, enzymology and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Controlled CXCL10/IP-10 expression in non-pathogenic SIVinfection. A. IP-10 gene expression levels in enriched lymph node CD4+cells are shown here as heatmap. Gene expression levels were assessedusing microarrays at different time points before and after SIVagm andSIVmac infection in AGM and MAC, respectively. B. IP-10 expressionlevels in plasma before and after SIVagm and SIVmac infection arerepresented here. Levels were quantified using a commercial ELISA (R&D)as described in (Liovat et al., PLoS One, 7(10): e46143, 2012). Medianfrom 12 AGM is displayed along with levels obtained in 3 MAC with arapid disease progression profile or with 2 MAC with a slow diseaseprogression profile. In addition data are shown also as a fold change oflevels measured at day 65 p.i. against median of values assessed beforeinfection for each animal. S=MAC slow progressor, P=MAC progressor andR=MAC rapid progressor.

FIG. 2: IP-10 robustness of prediction for rapid disease progression.Multivariate regression analysis. Only IP-10 was significant in themultivariate analysis for prediction of rapid disease progression, whileCD4⁺ T cell counts, plasma viral RNA and cellular viral DNA levels werenot.

FIG. 3: Decreased levels of plasma IP-10 antagonist form and sDPPIV-likeactivity during acute infection in HIV-1 infected individualsprogressing rapidly to disease. Frozen plasmas collected on EDTA wereobtained from 134 HIV. patients during primary infection (Fiebig stageIII-IV) of the ANRS Primo Cohort Co6, and described in (Liovat et al.,PLoS One, 7(10): e46143, 2012). Plasma IP-10 antagonist form (short) ispresented as a ratio short (antagonist form): total IP-10. Total IP10concentrations were previously measured (Liovat et al., PLoS One, 7(10):e46143, 2012). We also monitored the bioactive sDPPIV titers in plasma,determined with a luminescence-based assay (Promega). Mann-Whitney testwas used to compare groups of patients. Preliminary data from 53patients are shown: HIV neg (n=5-16), slow progressors (SP, n=11),progressors (P, n=17), rapid progressors (RP, n=25).

FIG. 4: Dynamics of plasma total IP-10 and sDPPIV-like activity afterseroconversion in HIV-1 infected individuals. Frozen sera collected onEDTA were obtained from 134 HIV⁺ patients at 3 (M3), 6 (M6) or 9 (M9)months post alleged date of seroconversion or even before infection(Pre). Patients were enrolled in the Amsterdam cohort Studies onHIV/AIDS. Total IP10 concentrations were measured as previouslydescribed (Liovat et al., PLoS One, 7(10): e46143, 2012). We alsomonitored the bioactive sDPPIV titers in plasma, determined with aluminescence-based assay (Promega). Wilcoxon signed rank test was usedto compare time points.

FIG. 5: IP-10 and DPPIV gene expression levels in the gut ofSIV-infected MAC and AGM. We sampled gut tissue at the time point ofeuthanasia in SIVmac-infected MAC (n=6) and SIVagm-infected AGM (n=5)used in other research projects. These necropsies were performed at day65 p.i., when inflammatory profiles are already distinct between MAC andAGM. We harvested fragments of jejunum, ileum, colon and rectum. Gutintra-epithelial cells (IEC) and gut mucosal CD4⁺ and CD4^(neg) cellswere enriched after collagenase digestion, centrifugation on a Percollgradient followed by a CD4 enrichment using magnetic beads. Genetranscript expression was determined by RT-PCR using Taqman geneexpression assays. Relative expressions were normalized first against18sRNA. On the top panel, Mann-Whitney test and Wilcoxon signed-ranktest were used to compare compartments between species or within eachspecies, respectively. On bottom panel, correlations were determinedusing the Spearman test on normalized values obtained in the specifiedcell compartment enriched from the high intestine (jejunum/Ileum) ofeach animal against values obtained in the specified cell compartmentenriched from the rectum of each animal.

FIG. 6: IP-10 and DPPIV gene expression levels in gut mucosal CD4+ cellscorrelate with dynamics of IP-10 and DPPIV in the blood of SIV-infectedMAC and AGM. Plasma total IP-10 and soluble DPPIV activity were measuredas described in previous figure legends at day 65 post-SIV infection ofMAC and AGM. Values were normalized against values (median) obtained inplasma samples of the same animals before infection and were expressedas fold change to baseline (FC o baseline). We searched for anycorrelation between those plasma dynamics with the gene expressionlevels in the gut mucosal CD4⁺ compartment. Each individual dotrepresents paired values for each studied animal.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by a skill artisanin chemistry, biochemistry, cellular biology, molecular biology, andmedical sciences.

The term “AIDS”, as used herein, refers to the symptomatic phase of HIVinfection, and includes both Acquired Immune Deficiency Syndrome(commonly known as AIDS) and “ARC,” or AIDS-Related Complex. See AdlerM, et al, Brit. Med. J. 1987; 294: 1145-1147. The immunological andclinical manifestations of AIDS are known in the art and include, forexample, opportunistic infections and cancers resulting from immunedeficiency.

As used herein, “T-cell” refers to a group of white blood cells known asT-lymphocytes that play a central role in cell-mediated immunity. Thereare several subsets of T cells, each with a distinct function (i.e.helper, memory, regulatory, natural killer).

The term “CD4⁺ T cells”, as used herein, refers to a type of T cellsthat expresses the CD4 marker. Said CD4⁺ T cells are generally treatedas having a pre-defined role as helper T cells within the immune system.“CD4”, as used herein, refers to a cluster of differentiation 4, aglycoprotein expressed on the surface of T helper cells, monocytes,macrophages, and dendritic cells. CD4 is a co-receptor that assists theT cell receptor (TCR) with an antigen-presenting cell. Using its portionthat resides inside the T cell, CD4 amplifies the signal generated bythe TCR by recruiting an enzyme, known as the tyrosine kinase lck, whichis essential for activating many molecules involved in the signalingcascade of an activated T cell.

The term “CD8+ T cells”, as used herein, refers to a type of T cell thatexpresses the CD8 marker. “CD8”, as used herein, refers to cluster ofdifferentiation 8, a transmembrane glycoprotein that serves as aco-receptor for the T cell receptor (TCR) expressed in the cytotoxic Tcells implicated in the rejection of transplants and the destruction oftumor and virally infected cells.

As used herein, “T-cell function” means any activities which areinherent to a T-cell. T-cell function means any one of cytokinesecretion, (for example, IL-2), proliferation or survival.

As used herein, “T-cell survival” means, the ability of a T-cell topersist in a host organism.

As used herein, “proliferation” refers to a process by which a cellundergoes mitosis, or increases in number, size or content.

The term “controller”, as used herein, refers to a HIV infected subjectthat exhibits a decrease in HIV viral load after infection and thatmaintains said decreased viral load levels over time. A “controller”also refers to an HIV-1 infected subject who remains asymptomatic withnormal CD4+ T-cell counts and low or undetectable plasma viral loadsdespite having never been treated with antiretroviral medications. HIVcontrollers are capable of maintaining very low viral load levels, forexample, plasma HIV R A levels <2000 copies/mL in the absence ofantiretroviral therapy, measured three times over a period spanning atleast 12 months. The features of controllers as defined by the HIVController Consortium are: i) to maintain HIV RNA levels below 2000copies/mL, ii) no antiretroviral therapy for 1 year or longer and iii)episodes of viremia are acceptable as long as they represent theminority of all available determinations.

The term “decreased”, as used herein, refers to the level of a HIVdisease prognosis marker, e.g. sIP-10, of a subject at least 1-fold(e.g. 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000,10,000-fold or more) lower than its reference value. “Decreased”, as itrefers to the level of a HIV disease prognosis marker, e.g. sIP-10, of asubject, signifies also at least 5% lower (e.g. 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%), 95%), 99%), or 100%) than the level in the reference sampleor with respect to the reference value for said prognosis marker.

As used herein, “diagnosis” or “identifying a subject having” refers toa process of determining if an individual is afflicted with a disease orailment (e.g., HIV). HIV is diagnosed for example by detecting eitherthe presence of an HIV polypeptide, HIV nucleic acid, or a markerassociated with HIV.

The term “HIV”, as used herein, include HIV-1 and HIV-2 and SIV. “HIV-1”means the human immunodeficiency virus type-1. HIV-1 includes, but isnot limited to, extracellular virus particles and the forms of HIV-1associated with HIV-1 infected cells. HIV-1 is known to comprise atleast ten subtypes (A1, A2, A3, A4, B, C, D, E, PL F2, G, H, J and K).See Taylor B, et al, MEJM 2008; 359(18): 1965-1966. Subtype B has beenassociated with the HIV epidemic in homosexual men and intravenous drugusers worldwide. Most HIV-1 immunogens, laboratory adapted isolates,reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa,India, and China, areas where the incidence of new HIV infections ishigh, HIV-1 subtype B accounts for only a small minority of infections,and subtype HIV-1 C appears to be the most common infecting subtype.“HIV-2” means the human immunodeficiency virus type-2. HIV-2 includes,but is not limited to, extracellular virus particles and the forms ofHIV-2 associated with HIV-2 infected cells. HIV-2 is known to include atleast five subtypes (A, B, C, D, and E). The term “SIV” refers to simianimmunodeficiency virus which is an HIV-like virus that infects monkeys,chimpanzees, and other nonhuman primates. SIV includes, but is notlimited, to extracellular virus particles and the forms of SIVassociated with SIV infected cells.

As used herein, a “HIV disease” encompasses basically all thephysiological conditions that are undergone by an individual who hasbeen infected by a HIV virus, starting from the time of the virusinfection event until the date of the individual's death, irrespectiveof whether the individual's death is a direct or indirect consequence ofthe virus infection event. It is recalled that the infection of anindividual with a HIV virus causes a chronic disease state thatprogressively causes a reduction of the effectiveness of the immunesystem and leaves the HIV-infected individuals susceptible toopportunistic infections and tumors. Thus, a HIV disease encompasses theprimary infection (or acute infection) time period, the seroconversiontime period, the asymptomatic stage time period, the early- andmedium-stage of HIV symptomatic disease, as well as the late stage ofHIV-1 disease (also called AIDS).

As used herein, “identifying” as it refers to a subject that has acondition refers to the process of assessing a subject and determiningthat the subject has a condition, for example, is infected with HIV.

The term “increased”, as used herein, refers to the level of a HIVdisease prognosis marker, e.g. sIP-10, of a subject at least 1-fold(e.g. 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000,10,000-fold or more) greater than its reference value. “Increased”, asit refers to the level of a HIV disease prognosis marker, e.g. sIP-10,of a subject, signifies also at least 5% greater (e.g. 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%), 95%), 99%), or 100%) than the level in thereference sample or with respect to the reference value for saidprognosis marker.

As intended herein, the “level” of a HIV disease prognosis marker, e.g.sIP-10, consists of a quantitative value of the said prognosis marker ina sample, e.g. in a sample collected from an HIV-infected patient. Insome embodiments, the said quantitative value does not consist of anabsolute value that is actually measured, but rather consists of a finalvalue resulting from the taking into consideration of a signal to noiseratio occurring with the assay format used, and/or the taking intoconsideration of calibration reference values that are used to increasereproducibility of the measures of the level of a HIV disease marker,from assay-to-assay. In some embodiments, the “level” of a HIV diseaseprognosis marker, e.g. sIP-10, is expressed as arbitrary units, sincewhat is important is that the same kind of arbitrary units are compared(i) from assay-to-assay, or (ii) from one HIV-infected patient toothers, or (iii) from assays performed at distinct time periods for thesame patient, or (iv) between the HIV prognosis marker level measured ina patients sample and a predetermined reference value (which may also betermed a “cut-off” value herein).

As used herein, “monitoring disease progression” refers to a process ofdetermining the severity or stage of a disease in an individualafflicted with the disease or ailment (e.g., HIV).

As used herein, “prognosis” refers to a process of predicting theprobable course and outcome of a disease in an individual afflicted witha disease or ailment (e.g., HIV), or the likelihood of recovery of anindividual from a disease (e.g., HIV).

The term “reference value”, as used herein, refers to the expressionlevel of a HIV disease prognosis marker under consideration (e.g.sIP-10) in a reference sample. A “reference sample”, as used herein,means a sample obtained from subjects, preferably two or more subjects,known to be free of the disease or, alternatively, from the generalpopulation. The suitable reference expression levels of HIV diseaseprognosis marker can be determined by measuring the expression levels ofsaid HIV disease prognosis marker in several suitable subjects, and suchreference levels can be adjusted to specific subject populations. Thereference value or reference level can be an absolute value; a relativevalue; a value that has an upper or a lower limit; a range of values; anaverage value; a median value, a mean value, or a value as compared to aparticular control or baseline value. A reference value can be based onan individual sample value such as, for example, a value obtained from asample from the subject being tested, but at an earlier point in time.The reference value can be based on a large number of samples, such asfrom population of subjects of the chronological age matched group, orbased on a pool of samples including or excluding the sample to betested.

As used herein, the term “biological sample” or “sample” refers to awhole organism or a subset of its tissues, cells or component parts(e.g. blood vessel, including artery, vein and capillary, body fluids,including but not limited to blood, serum, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). “Biological sample” furtherrefers to a homogenate, lysate or extract prepared from a whole organismor a subset of its tissues, cells or component parts, or a fraction orportion thereof. Lastly, “biological sample” refers to a medium, such asa nutrient broth or gel in which an organism has been propagated, whichcontains cellular components, such as proteins or nucleic acidmolecules.

As used herein, “selecting” refers to the process of determining that anidentified subject will receive an agent to treat the occurrence of acondition (e.g., HIV). Selecting can be based on an individual'ssusceptibility to a particular disease or condition due to, for example,family history, lifestyle, age, ethnicity, or other factors.

A “subject” which may be subjected to the methodology described hereinmay be any of mammalian animals including human, dog, cat, cattle, goat,pig, swine, sheep and monkey. A human subject can be known as a patient.In one embodiment, “subject” or “subject in need” refers to a mammalthat is infected with HIV or is suspected of being infected with HIV orhas been diagnosed with HIV infection. As used herein, an “HIV infectedsubject” refers to a mammal that is infected with HIV or has beendiagnosed with HIV infection. A “control subject” refers to a mammalthat is not infected with HIV, and is not suspected of being diagnosedwith HIV.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating the symptoms of a disorder (e.g., HIVinfection) and/or symptoms associated therewith. It will be appreciatedthat, although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

As used herein “treating” a disease in a subject or “treating” a subjecthaving a disease refers to subjecting the subject to a pharmaceuticaltreatment, e.g., the administration of a drug, such that the extent ofthe disease is decreased or prevented. For example, treating results inthe reduction of at least one sign or symptom of the disease orcondition. Treatment includes (but is not limited to) administration ofa composition, such as a pharmaceutical composition, and may beperformed either prophylactically, or subsequent to the initiation of apathologic event. Treatment can require administration of an agentand/or treatment more than once.

Methods for Prognosing an HIV Disease

IP-10 is a CXC chemokine (NP_001556) which functions to recruitactivated and memory lymphocytes to sites of inflammation. The secretedbioactive form (after cleavage of the signal peptide) is a polypeptideof 77 residues (positions 22-98 of NP_001556; SEQ ID NO: 1), hereindesignated IP-10, which binds the CXCR3 receptor. IP-10 also exists in atruncated form, herein referred to as “short IP-10” or “sIP-10”, whichis obtained by the removal of the two N-terminal amino acids of thesecreted bioactive form (amino acids 24-98 of NP_001556; SEQ ID NO: 2).

In contrast to native IP-10, sIP-10 is endowed with antagonistproperties and repels activated and memory lymphocytes from sites ofinflammation.

IP-10 has previously been identified as a negative indicator for chronicHCV patients receiving the IFN/ribavirin treatment, i.e. high levels ofplasma IP-10 are predictive of the failure to respond to this therapy.These elevated IP-10 levels are mainly in the short antagonist form,preventing trafficking of CXCR3⁺ lymphocytes capable to control HCVreplication, to the liver, thus diminishing HCV control.

The present application discloses sIP-10 as a clinically importantnegative predictive marker for likelihood to progress rapidly towardsAIDS. The present inventors have found that, surprisingly, sIP-10antagonist levels are dramatically reduced upon HIV infection. Moreover,they were reduced to a higher level in the rapid progressors than incomparison to slow progressors. Indeed, the inventors have shown thatsIP-10 is a strong negative predictor of rapid disease progression.Thus, whereas good prognosis is associated with low levels of sIP-10during HCV infection (Liovat et al., PLoS One, 7(10): e46143, 2012), itis correlated with high sIP-10 levels in the case of HIV infection,which was unexpected.

This represents an important and medically useful discovery. Thisdiscovery enables the discrimination of patients, prior to treatment,into a group of patients that is likely not to develop clinical AIDSsymptoms and a group of patients that will spontaneously progresstowards AIDS and will thus require specific and targeted therapeutictreatment. Determining that a patient is likely to remain asymptomaticmay save them from expensive treatment with significant side effects.This diagnostic tool may also assist physicians in identifying patientswho are likely to progress to AIDS and thus may suggest those patientsrequire earlier or more aggressive treatment.

In a first aspect, the present invention relates to a method ofdetermining the likelihood that a subject identified as being infectedwith HIV will develop AIDS, said method comprising the steps of:

-   -   a) measuring the sIP-10 level in a sample of the said subject;        and    -   b) determining the likelihood that said subject will develop        AIDS based on the level of step a).

As shown herein, a negative correlation has been surprisingly found bythe inventors between the levels of sIP-10 and the likelihood that apatient will progress towards AIDS. The lower the sIP-10 levels, thehigher the likelihood that the patient will develop AIDS clinicalsymptoms.

In an embodiment of the present invention, the said method comprises aprior step of obtaining a biological sample from the said subject.

The present invention also provides a method for prognosing an HIVdisease in a subject identified as being infected with HIV, said methodcomprising the steps of:

-   -   a) measuring the sIP-10 level in a sample of the said subject;        and    -   b) prognosing the said disease based on the level of step a).

In an embodiment of the present invention, the method for prognosing anHIV disease in a subject infected with an HIV virus includes a priorstep of obtaining a biological sample from the said subject.

As explained above, sIP-10 competes with IP-10 for CXCR3 and hasantagonistic properties. It is thus informative to express the levels ofsIP-10 as a ratio of sIP-10 versus total IP-10, instead of as raw valuesof sIP-10 concentrations. As used herein, “total IP-10” refers to allthe IP-10 isoforms. In other words the total IP-10 in a sample refers toall the IP-10(22-98) and sIP-10(24-98) molecules present in the sample.

In fact, not only is SIP-10 a negative predictor of a rapid progressionof an HIV disease, the inventors have shown that the ratio of sIP-10 tototal IP-10 was decreased during acute HIV infection in rapidprogressors compared to the other HIV-infected patients and compared tohealthy subjects.

Therefore, in a preferred embodiment, the method of the inventioncomprises a further step of measuring the total IP-10 level in thesample. According to another preferred embodiment, the method of theinvention comprises a further step of calculating the ratio of sIP-10 tototal IP-10. According to a further preferred embodiment, the method ofthe invention comprises the steps of:

-   -   a) measuring the sIP-10 level in a biological sample of a        subject identified as being infected with HIV;    -   b) measuring the total IP-10 level in said sample of the said        subject;    -   c) calculating the ratio of sIP-10 to total IP-10; and    -   d) prognosing the said disease based on the ratio of step c).

According to a further preferred embodiment, the method of the inventioncomprises the steps of:

-   -   a) obtaining a biological sample from a subject identified as        being infected with HIV;    -   b) measuring the sIP-10 level in said sample of the said        subject;    -   c) measuring the total IP-10 level in said sample of the said        subject;    -   d) calculating the ratio of sIP-10 to total IP-10; and    -   e) prognosing the said disease based on the ratio of step d).

In an embodiment of the method of the invention, the level of sIP-10 orthe ratio of sIP-10 to total IP-10 is compared to a reference value. Thereference value corresponds for example to the value of the said levelor the said ratio in a healthy subject. According to the presentinvention, a decreased level or ratio is indicative of a bad prognosis,i.e. the subject presents a high likelihood of progressing rapidlytowards AIDS. In other words, the said subject has a low likelihood of along term survival. On the other hand, an increased or a stable level orratio is indicative of a good prognosis. In this case, the subjectdisplays a low likelihood of progressing rapidly towards AIDS. In otherwords, the said subject has a high likelihood of a long term survival.

HIV Infected Subjects

Following infection with HIV-1, the rate of clinical disease progressionvaries between individuals. Factors such as host susceptibility,genetics and immune function, health care and co-infections as well asviral genetic variability[may affect the rate of progression to thepoint of needing to take medication in order not to develop AIDS. It isthus important to be capable of identifying those who show a higher riskof progressing rapidly towards AIDS.

HIV disease staging and classification systems are critical toolsproviding clinicians and patients essential information for clinicalmanagement. The CDC disease staging system assesses the severity of theHIV disease by CD4⁺ T lymphocyte cell (CD4) counts and by the presenceof specific HIV-related conditions. This system describes the infectionin three stages:

-   -   Stage 1: T cell counts ≧500 cells/μl and no AIDS defining        conditions;    -   Stage 2: T cell counts 200 to 500 cells/μl and no AIDS defining        conditions; and    -   Stage 3: T cell counts ≦200 cells/μl or AIDS defining        conditions.

Thus in an embodiment of the method of the invention, the subject isselected in the group of subjects having T cell counts of less than 200cells/μL, comprised between 200 and 500 cells/μL, and above 500cells/μL. In another embodiment of the method of the invention, thesubject has T cell counts of less than 200 cells/μL. In anotherembodiment of the method of the invention, the subject has T cell countscomprised between 200 and 500 cells/μL. In another embodiment of themethod of the invention, the subject has T cell counts above 500cells/μL.

According to the CDC staging system, the definition of Al DS includesall HIV-infected individuals with CD4 counts of less than 200 cells/μL,or a CD4 percentage (over all lymphocytes) of less than 14%, as well asthose with certain HIV-related conditions and symptoms. Furthermore, theCDC staging system classifies as eligible for antiretroviral therapysubjects with CD4⁺ T-lymphocyte counts of less than 500 cells/μL. Yet,some persons having T cell counts above 500 cells/μL are rapidprogressors who will advance towards AIDS in the next few months. Thosepeople may also benefit from antiretroviral therapy. It would thus bebeneficial to be capable of identifying those subjects which still haveT cell counts above 500 cells/μL but have a high risk of developing AIDSin the near future.

Thus in another embodiment, the method of the invention relates to amethod for determining the likelihood that a subject identified as beinginfected with HIV and having T cell counts above 500 cells/μL willdevelop AIDS, said method comprising the steps of:

-   -   a) measuring the sIP-10 level in a sample of the said subject;        and    -   b) determining the said likelihood based on the level of step        a).

In another aspect of the present invention, a method is provided fordetermining the likelihood that a subject identified as being infectedwith HIV and having T cell counts above 500 cells/μL will develop AIDS.Generally, the method includes at least the following steps:

-   -   a) obtaining a biological sample from a subject identified as        being infected with HIV and having T cell counts above 500        cells/μL;    -   b) measuring the sIP-10 level in said sample of the said        subject; and    -   c) determining the said likelihood based on the level of step        b).

In another embodiment, the method of the invention relates to a methodof prognosing an HIV disease in a subject identified as being infectedwith HIV and having T cell counts above 500 cells/μL, said methodcomprising the steps of:

-   -   a) measuring the sIP-10 level in a sample of the said subject;        and    -   b) prognosing the said disease based on the level of step a).

In another aspect of the present invention, a method is provided forprognosing an HIV disease in a subject infected with an HIV virus andhaving T cell counts above 500 cells/μL. Generally, the method includesat least the following steps:

-   -   a) obtaining a biological sample from a subject identified as        being infected with HIV and having T cell counts above 500        cells/μL;    -   b) measuring the sIP-10 level in said sample of the said        subject; and    -   c) prognosing the said disease based on the level of step b).

In a preferred embodiment, the method of the invention comprises afurther step of measuring the total IP-10 level in the sample. Accordingto another preferred embodiment, the method of the invention comprises afurther step of calculating the ratio of sIP-10 to total IP-10.According to a further preferred embodiment, the method of the inventioncomprises the steps of:

-   -   a) measuring the sIP-10 level in a biological sample from a        subject identified as being infected with HIV and having T cell        counts above 500 cells/μL;    -   b) measuring the total IP-10 level in said sample of the said        subject;    -   c) calculating the ratio of sIP-10 to total IP-10; and    -   d) prognosing the said disease based on the ratio of step c).

According to a further preferred embodiment, the method of the inventioncomprises the steps of:

-   -   a) obtaining a biological sample from a subject identified as        being infected with HIV and having T cell counts above 500        cells/μL;    -   b) measuring the sIP-10 level in said sample of the said        subject;    -   c) measuring the total IP-10 level in said sample of the said        subject;    -   d) calculating the ratio of sIP-10 to total IP-10; and    -   e) prognosing the said disease based on the ratio of step d).

In an embodiment of the method of the invention, the level of sIP-10 orthe ratio of sIP-10 to total IP-10 is compared to a reference value. Thereference value corresponds for example to the value of the said levelor the said ratio in a healthy subject. According to the presentinvention, a decreased level or ratio is indicative of a bad prognosis,i.e. the subject presents a high likelihood of progressing rapidlytowards AIDS. In other words, the said subject has a low likelihood of along term survival. On the other hand, an increased or a stable level orratio is indicative of a good prognosis. In this case, the subjectdisplays a low likelihood of progressing rapidly towards AIDS. In otherwords, the said subject has a high likelihood of a long term survival.

Human dipeptidyl peptidase IV (DPPIV), which is also known as CD26, is a110 kDa cell surface molecule. The amino acid sequence of human DPPIVprotein comprises 766 amino acids and is shown in SEQ ID NO: 3(Swissprot database Accession No. P27487). It contains intrinsicdipeptidyl peptidase IV activity which selectively removes N-terminaldipeptide from peptides with proline or alanine in the third amino acidposition. It interacts with various extracellular molecules and is alsoinvolved in intracellular signal transduction cascades. Themultifunctional activities of human DPPIV are dependent on cell type andintracellular or extracellular conditions that influence its role as aproteolytic enzyme, cell surface receptor, co-stimulatory interactingprotein and signal transduction mediator. Human DPPIV has a shortcytoplasmatic domain from amino acid position 1 to 6, a transmembraneregion from amino acid position 7 to 28, and an extracellular domainfrom amino acid position 29 to 766 with intrinsic dipeptidyl peptidaseIV (DPPIV) activity.

It has been shown in the literature that IP-10 is cleaved by DPPIV,resulting in the generation of a short form of IP-10 (sIP-10) that actsas an antagonist of CXCR3 (De Meester et al, 1999, Immunol Today, 20(8):367-375).

The inventors have shown that plasma DPPIV activity is reduced duringacute infection in those HIV-infected patients who subsequentlyprogressed more rapidly than the other patients towards AIDS, just likethe level of sIP-10. In other words, the activity of DPPIV can be usedas a convenient proxy for the level of SIP-10.

The present invention thus also relates to a method of determining thelikelihood that a subject identified as being infected with HIV willdevelop AIDS, said method comprising the steps of:

-   -   a) measuring the DPPIV activity in a sample of the said subject;        and    -   b) determining the likelihood that said subject will develop        AIDS based on the level of step a).

In an embodiment of the present invention, the said method comprises aprior step of obtaining a biological sample from the said subject.

The present invention also provides a method for prognosing an HIVdisease in a subject identified as being infected with HIV, said methodcomprising the steps of:

-   -   a) measuring the DPPIV activity in a sample of the said subject;        and    -   b) prognosing the said disease based on the level of step a).

In an embodiment of the present invention, the method for prognosing anHIV disease in a subject infected with an HIV virus includes a priorstep of obtaining a biological sample from the said subject.

In an embodiment of the method of the invention, the DPPIV activity iscompared to a reference value. The reference value corresponds forexample to the value of the said activity in a healthy subject.According to the present invention, a decreased activity is indicativeof a bad prognosis, i.e. the subject presents a high likelihood ofprogressing rapidly towards AIDS. In other words, the said subject has alow likelihood of a long term survival. On the other hand, an increasedor a stable activity is indicative of a good prognosis. In this case,the subject displays a low likelihood of progressing rapidly towardsAIDS. In other words, the said subject has a high likelihood of a longterm survival.

In another aspect, the method of the invention relates to a method ofprognosing an HIV disease in a subject identified as being infected withHIV and having T cell counts above 500 cells/μL, said method comprisingthe steps of:

-   -   a) measuring the DPPIV activity in a sample of the said subject;        and    -   b) prognosing the said disease based on the activity of step a).

In an embodiment of the present invention, the said method comprises aprior step of obtaining a biological sample from the said subject.

In an embodiment of the method of the invention, the activity of DPPIVis compared to a reference value. The reference value corresponds forexample to the value of the said activity in a healthy subject.According to the present invention, a decreased activity is indicativeof a bad prognosis, i.e. the subject presents a high likelihood ofprogressing rapidly towards AIDS. In other words, the said subject has alow likelihood of a long term survival. On the other hand, an increasedor a stable activity is indicative of a good prognosis. In this case,the subject displays a low likelihood of progressing rapidly towardsAIDS. In other words, the said subject has a high likelihood of a longterm survival.

For the methods herein, the biological sample is preferably a samplefrom blood, plasma, lymph, bone marrow fluid, pleural fluid, peritonealfluid, spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinalfluid, brain fluid, ascites, urine, saliva, bronchial lavage, bile,sweat, tears, ear flow, sputum, semen, vaginal flow, milk, amnioticfluid, or secretions of respiratory, intestinal or genitourinary tract.Exemplary of such samples, is a peripheral blood sample, and a bodyfluid sample that contains dissociated bone marrow cells from a bonemarrow biopsy. The volume of sample is any that is convenient fortesting, such as at least about 0.01 mL to about 50 mL or 100 ml.

The preparation and/or isolation of proteinaceous material from a samplefor analysis are also well known in this field.

Antibodies for sIP-10 and IP-10

Measuring the levels of sIP-10 expression in a sample is easilyundertaken by one knowledgeable in this field. Non-limiting examplesinclude western-blot analysis, ELISA assays, immuno-reactivity assays,column assays using fluorescence, antibodies and radiolabeled markers.Automated manners of performing the analysis, for example, using acomputer processor can also be used.

Total, long and short (truncated) IP-10 can be determined utilizingpolyclonal (rabbit) and monoclonal (mouse) antibodies that are specificfor the long form of IP-10. In an added embodiment, in vivo IP-10activity can be assessed by using CXCR3 cells as a surrogate for IP-10activity. Flow cytometry can be used to measure the relative amount ofCXCR3 cells in the peripheral blood of patients throughout theirtreatment. This will serve as a reflection of the ability of activated Tcells and B cells to respond to a gradient of IP-10 (keeping in mindthat there exist other CXCR3 agonists). In another added embodiment, adipeptidyl peptidase IV activity can be measured in patient plasma,which can correlate to the IP-10 cleavage.

In this analysis, it is advantageous to inactivate the plasma DPPIVactivity, thus preventing an extra-corporeal cleavage of IP-10.Collections are thus preferably performed with protease inhibitors. Forexample, one such commercially available sample collection tube is BD™P700 but other such BD™ 100 are also sufficient to preserve IP-10 in itscirculating form.

In one embodiment, antibodies specific for the long form of IP-10 toevaluate the amount of long vs. short form of IP-10 are generated. Suchantibodies that distinguish full-length IP-10 from short (truncated)form of IP-10, were generated by immunization of rabbits with asynthetic peptide coupled to KLH; (VPLSRTVRC (positions 22-30 ofNP_001556; SEQ ID NO: 4) corresponding of the NH₂ terminal of thefull-length IP-10 protein). Immunoglobulins G (IgG) can then be purifiedin two steps by chromatography affinity: the first step is a positiveselection of IgG that recognize the long peptide. The second step isdepletion of IgG that recognize the shorter peptide LSRTVRC (SEQ ID NO:4) common to short and full-length form of IP-10. Purified antibodiesare specific to full-length IP-10. The same strategy can be used toobtain polyclonal antibodies from different species (e.g., goat,chicken, and others). Monoclonal antibodies may also be derived frommice after immunization with the long peptide. After verifying thepresence of antibody production in the serum, hybridomas are producedand cloned. The hybridoma screening can be made by direct ELISA withboth, the long and short peptides, and the full-length and short form ofrecombinant IP-10 coated directly in ELISA plate. Then antibodies fromhybridomas specific of the full-length can be purified. Validation ofpolyclonal antibodies can be performed by direct and by sandwiches Elisaassays against short and full-length IP-10 form. SIP-10 can be generatedby following in vitro digestion of recombinant full-length IP-10 byrecombinant DPIV. The two NH₂ amino-acids, Valine and Proline aretruncated from the full-length. Monoclonal antibodies discriminatingbetween the short and the long forms of IP-10 have been described ine.g. U.S. Pat. No. 8,124,332 and in Casrouge et al., J Clin Invest.,121(1): 308-317, 2011.

sIP-10 Levels Assay

Using these distinguishing antibodies, IP-10 can be detected inbiological fluids, for example, using an ELISA assay to specificallydetect the full-length IP-10. As IP-10 binds to heparin, heparincross-linked to agarose beads via amide bonds can be used to concentrateIP-10 from plasma. IP-10 can be eluted with NaCl 1M. To avoid or atleast minimize ex vivo truncation of IP-10 in biologic samples,inhibitors of DPIV can be added into collection tubes. Various DPIVinhibitors are known in the art, e.g., a large number of DPIV inhibitorshave been described and their structures and characteristics have beensuccinctly reviewed (see e.g. U.S. Pat. No. 4,935,493; U.S. Pat. No.5,462,928; U.S. Pat. No. 5,543,396; U.S. Pat. No. 5,296,604; U.S. Pat.No. 6,100,234; PCT/US92/09845; Augustyns et al., Curr Med Chem., 6(4):311-327, 1999; Evans, IDrugs, 5: 577-585, 2002; Weber, J Med Chem.,47(17): 4135-4141, 2004; McIntosh et al., Int J Biochem & Cell Biol,38(5-6): 860-872, 2006; Wiedeman & Trevillyan, Curr Opin Investig Drugs,4(4): 412-420; 2003). The HuCAl (Human Combinatorial Antibody library)technology allows to generate in vitro a 1.5 billion human antibodiescandidates library. An automated panning process is used: the peptidesspecific of long and short forms of IP-10 are immobilized in 384-wellmicroplate for screening against antibody-displaying phage library.Specificity of antibodies is then verified by ELISA.

To generate the short (truncated) IP-10 form, the full length IP-10sequence(VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKEMSKRSP,SEQ ID NO: 1) can be incubated with DPIV at 25-37° C. to generate theshort form(LSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKEMSKRSP,SEQ ID NO: 2). DPIV is a known protein, with a cloned sequence (e.g.,from mammals such as human and mouse). Resolution of IP-10 and sIP-10can be accomplished by Western blotting or by MALDI-TOF-MS as is knownin the art.

In other embodiments, the antibodies that distinguish between the shortand long forms of IP-10 can be used to evaluate patients with chronicinflammation (e.g., cancer, obesity, autoimmunity, graft vs. hostdisease). Specific applications can be focused on diseases in whichIP-10 and/or DPIV have been shown to be elevated (e.g., melanoma, typeII diabetes, autoimmune vasculitis).

Soluble DPPIV Expression

Human soluble dipeptidyl peptidase IV (soluble DPPIV) amino acidsequence is shown in SEQ ID NO: 3, and comprises the amino acidpositions 29 to 766 from Swissprot database Accession number P27487. Thedimer of soluble DPPIV is a 170 kDa glycoprotein consisting of twoidentical monomeric soluble DPPIV units.

To express a soluble DPPIV enzyme, the polynucleotide can be insertedinto an expression vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing sequences encoding a solubleDPPIV polypeptide and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such techniques are described, for example, in Sambrook et al. (1989)and in Ausubel et al (1989).

A variety of expression vector/host systems can be utilized to containand express sequences encoding a soluble DPPIV polypeptide. Theseinclude, but are not limited to, microorganisms, such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors,insect cell systems infected with virus “expression vectors (e.g.,baculovirus), plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids),or animal cell systems.

The control elements or regulatory sequences are those nontranslatedregions of the vector enhancers, promoters, 5′ and 3′ untranslatedregions which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.)or pSPORTI plasmid (Life Technologies) and the like can be used. Thebaculovirus polyhedrin promoter can be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) can be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding a solubleDPPIV enzyme polypeptide, vectors based on SV40 or EBV can be used withan appropriate selectable marker.

DPPIV Assay

Assays for DPPIV activity are well known in the art. DPPIV activity canbe assayed using a substrate which reacts with DPPIV to form adetectable product, as described in U.S. Pat. No. 5,601,986. Suitableenzyme substrates include, but are not limited to, dipeptide substratessuch as Xaa-pro-para-nitro-analide (Xaa-Pro-PNA) or Xaa-Pro-coumarin.The variable amino acid, Xaa, can be any naturally occurring orsynthetic amino acid. An exemplary dipeptide substrate isGly-Pro-para-nitro-analide (Gly-Pro-PNA). At a wavelength of 405nanometers, the substrate has no absorbance; however, if the dipeptidesubstrate is cleaved (after the Pro) due to the presence of DPPIV, theformation of a reaction product can be visualizedspectrophotometrically, as a yellow-green color is produced. Othersubstrates, such as Xaa-Pro-coumarin, can be visualizedspectrofluorometrically as a fluorescent emission is produced by thereaction.

DPPIV assays embodying such reagents and reactions can be performed inany suitable reaction vessel, for example, a test tube or well of amicrotiter plate. Enzyme activities typically are measured at 24° C., bymixing 50 or 100 μl of enzyme sample to 100 or 150 μl microliters of areaction buffer containing 200 μM of chromogenic substrate, such asGly-Pro-PNA (commercially available from Bachem, San Diego, Calif.) in0.1 M Tris-HCl buffered Triton X-100 (0.1% v/v) at pH 7.0. The reactionsare incubated for 30 minutes, and optical density readings are taken at405 nm. During the reaction time course, several optical densityreadings are taken at different time points. DPPIV enzyme activity isexpressed in nmol/min/ml based on the progression curve calculated fromthe concentration of hydrolyzed substrates.

Methods of Treatment

A subject identified as being infected with HIV and who has a badprognosis would benefit from being treated with an anti-HIV therapybefore the appearance of the AIDS symptoms.

A practitioner treats HIV infection by taking actions to ameliorate thecauses or symptoms of the infection in a patient. Treatment of HIVcomprises administering therapy to a patient. Therapy may include:selecting and administering one or more anti-HIV drugs to the patient,adjusting the dosage of the anti-HIV drug, adjusting the dosing scheduleof the drug, and adjusting the length of the therapy. Anti-HIV drugs areselected by practitioners based on the nature of the infection, thepatient's response to the infection and the patient's response to thedrug. The dosage of the anti-HIV drug can be adjusted as well by thepractitioner based on the nature of the drug, the nature of theinfection, the patient's response to the infection, and the patient'sresponse to the drug. The dosing schedule can also be adjusted by thepractitioner based on the nature of the drug, the nature of theinfection, the patient's response to the infection, and the patient'sresponse to the drug. Also, the length of the therapy can be adjusted bythe practitioner based on the nature of the drug, the nature of theinfection, the patient's response to the infection, the patient'sresponse to the drug. Also, the practitioner can select between a singledrug therapy, a dual drug therapy, or a triple drug therapy. Also, theanti-HIV therapy can be adjusted by the practitioner based on whetherthe patient suffers from acute HIV infection, chronic HIV infection orAIDS.

Antiretroviral or anti-HIV disease therapy can include, but is notlimited to, highly active antiretroviral therapy (HAART), proteaseinhibitors, fusion inhibitors, integrase inhibitors, co-receptorspecific agents, 3TC, AZT, nevirapine, non-nucleoside analogue reversetranscriptase inhibitors and nucleoside analogue reverse transcriptaseinhibitors. HAART can be three or more antiretroviral drugs incombination, including at least one protease inhibitor, or at least areverse transcriptase inhibitor and a protease inhibitor; or at leasttwo reverse transcriptase inhibitors with at least one proteaseinhibitor.

Typical reverse transcriptase inhibitors include nucleoside analogs,e.g., AZT (Zidovudine), ddi (didanosine), ddc (zalcitabine), D4T(stavudine), 3TC (lamivudine), Ziagen (abacavir), combivir (mix of AZTand 3TC), and non-nucleoside analogs, e.g., viramune (nevirapine),rescriptor (delavirdine), sustiva (efavirenz). Protease inhibitorsinclude invirase (saquinavir), norvir (ritonavir), crixivan (indinavir),viracept (nelfinavir), agenerase (amprenivir), kaletra (lopinavir andritonavir) and fortovase (saquinavir in a soft gelatin form). Thus,HAART can also be “triple cocktail” therapy. That is, a three drugregimen is used to combat HIV wherein one of the three drugs is usuallya protease inhibitor (and the other two are usually reversetranscriptase inhibitors).

In a particular aspect, the invention relates to a method for selectingan anti-HIV therapy for a subject identified as being infected with HIV.Generally, the method of the invention comprises at least the followingsteps:

-   -   a) determining the likelihood that said subject will develop        AIDS by any one of the above methods; and    -   b) selecting the therapy based on the said likelihood.

In one embodiment the invention relates to a method for selecting ananti-HIV therapy for a subject identified as being infected with HIV.Generally, the method of the invention comprises at least the followingsteps:

-   -   a) determining the prognosis of the said subject by any one of        the above methods; and    -   b) selecting the therapy based on the said prognosis.

In one embodiment the therapy is selected from the group consisting of:highly active antiretroviral therapy (HAART), protease inhibitors,fusion inhibitors, integrase inhibitors, co-receptor specific agents,3TC, AZT, nevirapine, non-nucleoside analogue reverse transcriptaseinhibitors, nucleoside analogue reverse transcriptase inhibitors andvaccine therapy.

HIV-infected subjects who efficiently control viral replication becausethey are under anti-HIV therapy, e.g. HAART regimen, still display lowlevels of chronic inflammation. HIV-infected subjects, who interruptantiretroviral treatment, generally display a strong viral rebound.During this viral rebound, the subjects are highly transmissible forHIV. Interestingly, between 5 and 15% of HIV-infected subjects whostarted HIV therapy, e.g., HAART, early, spontaneously control viralreplication after treatment interruption. More generally, it would beadvantageous to be capable of monitoring the efficacy of anti-HIVtherapy for subjects identified as being infected with HIV. Inparticular, it would thus be useful to be capable of identifying thesubjects who have a high likelihood of controlling viral replication andnot developing AIDS. These subjects would thus benefit from arrestingtheir treatment. Alternatively, it would be useful to identify thesubjects who have a high likelihood of progressing towards AIDS if theirtreatment was stopped. These patients should be maintained on an HIVtherapy.

The present invention thus provides a method for monitoring the efficacyof an anti-HIV therapy for a subject identified as being infected withHIV, said method comprising the steps of:

-   -   a) determining the likelihood that said subject will develop        AIDS by any one of the above methods; and    -   b) assessing the efficacy of said therapy based on the        determination of step a).

Efficacy of HIV treatment can be determined using the methods providedherein. In particular examples, the level of sIP-10 or the ratio ofsIP-10 to total IP-10 or the activity of DPPIV is measured at a firsttime point using the methods provided and then compared to the level ofsIP-10 or the ratio of sIP-10 to total IP-10 or the activity of DPPIV,respectively, measured at a second later time point by the same method.In some examples, the first time point is at a predetermined time priorto administration of a therapy, such as an anti-HIV therapy, and thesecond time point is at a predetermined time following administration ofthe therapy, during the administration of the therapy, or betweensuccessive administrations of the therapy. In exemplary methods, thesample can be obtained from the subject, for example, at least, at aboutor at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20weeks, or later following administration of the anti-HIV therapy to thesubject. In some examples, samples are collected at a plurality of timepoints, such as at more than one time point, including, for example, at1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more time points followingadministration of the anti-HIV therapy to the subject. In some examples,samples are collected at regular intervals following administration ofthe anti-HIV therapy to the subject.

In particular examples, the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV is measured at a first time andthen compared to the level of sIP-10 or the ratio of sIP-10 to totalIP-10 or the activity of DPPIV, respectively, measured at a second latertime point to determine the likelihood of developing AIDS over time,where if the level of sIP-10 or the ratio of sIP-10 to total IP-10 orthe activity of DPPIV at the second time point is more than the level ofsIP-10 or the ratio of sIP-10 to total IP-10 or the activity of DPPIV,respectively, at the first time point, then the likelihood that thesubject will develop AIDS has decreased. In particular examples, if thelevel of sIP-10 or the ratio of sIP-10 to total IP-10 or the activity ofDPPIV at a second time point is 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 or more times greater than the level of sIP-10 or the ratio ofsIP-10 to total IP-10 or the activity of DPPIV, respectively, at a firsttime point, then the likelihood that the subject will develop AIDS hasdecreased.

In particular examples, the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV is measured at a first time andthen compared to the level of sIP-10 or the ratio of sIP-10 to totalIP-10 or the activity of DPPIV, respectively, measured at a second latertime point to determine stabilization of the likelihood to progresstowards AIDS over time, where if the level of sIP-10 or the ratio ofsIP-10 to total IP-10 or the activity of DPPIV at the second time pointis equal to or about the same as the level of sIP-10 or the ratio ofsIP-10 to total IP-10 or the activity of DPPIV at the first time point,then the likelihood to progress towards AIDS has stabilized.

In particular examples, the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV is measured at a first time pointand then compared to the level of sIP-10 or the ratio of sIP-10 to totalIP-10 or the activity of DPPIV, respectively, measured at a second latertime point to determine the effectiveness of therapy in inhibiting HIVdisease progression, where if the level of sIP-10 or the ratio of sIP-10to total IP-10 or the activity of DPPIV at the second time point is morethan or equal to the level of sIP-10 or the ratio of sIP-10 to totalIP-10 or the activity of DPPIV, respectively, at the first time point,then the therapy is effective at inhibiting HIV disease progression. Inparticular examples, if the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV at a second time point is equal toor 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or more times greater thanthe level of sIP-10 or the ratio of sIP-10 to total IP-10 or theactivity of DPPIV, respectively, at a first time point, then the therapyis effective at inhibiting HIV disease progression.

In particular examples, the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV is measured at a first time pointand then compared to the level of sIP-10 or the ratio of sIP-10 to totalIP-10 or the activity of DPPIV, respectively, measured at a second latertime point to determine the effectiveness of therapy in inhibiting HIVdisease progression, where if the level of sIP-10 or the ratio of sIP-10to total IP-10 or the activity of DPPIV at the first time point isgreater than the level of sIP-10 or the ratio of sIP-10 to total IP-10or the activity of DPPIV, respectively, at the second time point, thenthe therapy is not effective at inhibiting HIV disease progression. Inparticular examples, if the level of sIP-10 or the ratio of sIP-10 tototal IP-10 or the activity of DPPIV at a first time point is 2, 3, 4,5, 6, 7, 8, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000 or more times greater than the levelof sIP-10 or the ratio of sIP-10 to total IP-10 or the activity ofDPPIV, respectively, at a second time point, then the therapy is noteffective at inhibiting HIV disease progression.

In some examples, the methods provided herein can detect at or about a2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold,900-fold, 1000-fold or higher increase in the level of sIP-10 or theratio of sIP-10 to total IP-10 or the activity of DPPIV over timerelative to a control sample. in particular examples, the methodsprovided herein can detect at or about a 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 200-fold, 300-fold, 400-fold,500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or higherdecrease in the level of sIP-10 or the ratio of sIP-10 to total IP-10 orthe activity of DPPIV over time relative to a control sample. In someexamples, the control sample is a sample obtained from a subject at afirst time point and compared to a sample obtained from the subject at asecond time point. In some examples, the control sample is a sample witha known level of sIP-10 or ratio of sIP-10 to total IP-10 or activity ofDPPIV. In some examples, the control sample is a sample obtained from asubject with a particular HIV disease, a known stage of HIV disease, ora known HIV disease prognosis.

The present invention further relates to a method for adjusting a HIVtherapy for a subject identified as being infected with HIV, said methodcomprising the steps of:

-   -   a) assessing the efficacy of an anti-HIV therapy for said        subject by any one of the above methods; and    -   b) adapting the said treatment based on said assessment.

Said adaptation of the HIV therapy may consist in:

-   -   a reduction or suppression of the said HIV therapy if the        therapy is assessed as being effective, or    -   the continuation or an augmentation of the said HIV therapy if        the therapy is assessed as not being effective.

In one embodiment, the practitioner adjusts the therapy based on thepatient's level of sIP-10 or ratio of sIP-10 to total IP-10 or activityof DPPIV compared to a reference level. In one embodiment, thepractitioner adjusts the therapy by selecting and administering adifferent drug. In one embodiment, the practitioner adjusts the therapyby selecting and administering a different combination of drugs. In oneembodiment, the practitioner adjusts the therapy by adjusting drugdosage. In one embodiment, the practitioner adjusts the therapy byadjusting dose schedule. In one embodiment, the practitioner adjusts thetherapy by adjusting length of therapy. In one embodiment, thepractitioner adjusts the therapy by selecting and administering adifferent drug combination and adjusting drug dosage. In one embodiment,the practitioner adjusts the therapy by selecting and administering adifferent drug combination and adjusting dose schedule. In oneembodiment, the practitioner adjusts the therapy by selecting andadministering a different drug combination and adjusting length oftherapy. In one embodiment, the practitioner adjusts the therapy byadjusting drug dosage and dose schedule. In one embodiment, thepractitioner adjusts the therapy by adjusting drug dosage and adjustinglength of therapy. In one embodiment, the practitioner adjusts thetherapy by adjusting dose schedule and adjusting length of therapy. Inone embodiment, the practitioner adjusts the therapy by selecting andadministering a different drug, adjusting drug dosage, and adjustingdose schedule. In one embodiment, the practitioner adjusts the therapyby selecting and administering a different drug, adjusting drug dosage,and adjusting length of therapy. In one embodiment, the practitioneradjusts the therapy by selecting and administering a different drug,adjusting dose schedule, and adjusting length of therapy. In oneembodiment, the practitioner adjusts the therapy by adjusting drugdosage, adjusting dose schedule, and adjusting length of therapy. In oneembodiment, the practitioner adjusts the therapy by selecting andadministering a different drug, adjusting drug dosage, adjusting doseschedule, and adjusting length of therapy.

In one embodiment, therapy comprises the selection and administration ofan anti-HIV drug to the patient by the practitioner. In one embodiment,the anti-HIV disease drug comprises protease inhibitors. In oneembodiment, the anti-HIV disease drug comprises fusion inhibitors. Inone embodiment, the anti-HIV disease drug comprises integraseinhibitors. In one embodiment, the anti-HIV disease drug comprisesco-receptor specific agents. In one embodiment, the anti-HIV diseasedrug comprises 3TC. In one embodiment, the anti-HIV disease drugantiviral interferon comprises AZT. In one embodiment, the anti-HIVdisease drug comprises polymerase inhibitor. In one embodiment, theanti-HIV disease drug comprises protease inhibitor. In one embodiment,the anti-HCV drug comprises nevirapine. In one embodiment, the anti-HIVdisease drug comprises non-nucleoside analogue reverse transcriptaseinhibitors. In one embodiment, the anti-HIV disease drug comprisesnucleoside analogue reverse transcriptase inhibitors.

In another embodiment, therapy comprises the selection andadministration of two anti-HIV disease drugs to the patient by thepractitioner as part of dual therapy. In one embodiment the two dualtherapy drugs are protease inhibitors. In one embodiment the two dualtherapy drugs are non-nucleoside analogue reverse transcriptaseinhibitors. In one embodiment the two dual therapy drugs are nucleosideanalogue reverse transcriptase inhibitors. In one embodiment the twodual therapy drugs are a protease inhibitor and a non-nucleosideanalogue reverse transcriptase inhibitor. In one embodiment the two dualtherapy drugs are a protease inhibitor and a nucleoside analogue reversetranscriptase inhibitor. In one embodiment the two dual therapy drugsare a non-nucleoside analogue reverse transcriptase inhibitor andnucleoside analogue reverse transcriptase inhibitor.

In another embodiment, therapy comprises the selection andadministration of three anti-HIV disease drugs to the patient by thepractitioner as part of triple therapy. In one embodiment the threetriple therapy drugs are an interferon drug, ribavirin, and a NS3protease inhibitor. In one embodiment, the triple therapy drugs comprisehighly active antiretroviral therapy (HAART). In one embodiment, theanti-HIV disease drug comprises HAART, said HAART being three or moreantiretroviral drugs in combination, including at least one proteaseinhibitor. In one embodiment, the anti-HIV disease drug comprises HAART,said HAART being three or more antiretroviral drugs in combination,including at least a reverse transcriptase inhibitor and a proteaseinhibitor. In one embodiment, the anti-HIV disease drug comprises HAART,said HAART being three or more antiretroviral drugs in combination,including at least two reverse transcriptase inhibitors with at leastone protease inhibitor.

In one embodiment where there is an decreased level of level of sIP-10or ratio of sIP-10 to total IP-10 or activity of DPPIV with respect to areference level, treatment comprises a less aggressive therapy than areference therapy. In one embodiment a less aggressive therapy comprisesnot administering drugs and taking a “watchful waiting” approach. In oneembodiment a less aggressive therapy comprises delaying administrationof anti-HIV disease drugs. In one embodiment a less aggressive therapycomprises selecting and administering less potent drugs. In oneembodiment a less aggressive therapy comprises decreasing dosage ofanti-HIV disease drugs. In one embodiment a less aggressive therapycomprises decreasing the frequency of the dose schedule. In oneembodiment a less aggressive therapy comprises shortening length oftherapy. In one embodiment, less aggressive therapy comprises selectingand administering less potent drugs and decreasing drug dosage. In oneembodiment, less aggressive therapy comprises selecting andadministering less potent drugs and decreasing dose schedule. In oneembodiment, less aggressive therapy comprises selecting andadministering less potent drugs and shortening length of therapy. In oneembodiment, less aggressive therapy comprises decreasing drug dosage anddecreasing dose schedule. In one embodiment, less aggressive therapycomprises decreasing drug dosage and shortening length of therapy. Inone embodiment, less aggressive therapy comprises decreasing doseschedule and shortening length of therapy. In one embodiment, lessaggressive therapy comprises selecting and administering less potentdrugs, decreasing drug dosage, and decreasing dose schedule. In oneembodiment, less aggressive therapy comprises selecting andadministering less potent drugs, decreasing drug dosage, and shorteninglength of therapy. In one embodiment, less aggressive therapy comprisesselecting and administering less potent drugs, decreasing dose schedule,and shortening length of therapy. In one embodiment, less aggressivetherapy comprises decreasing drug dosage, decreasing dose schedule, andshortening length of therapy. In one embodiment, less aggressive therapycomprises selecting and administering less potent drugs, decreasing drugdosage, decreasing dose schedule, and shortening length of therapy.

In one embodiment a less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy. In oneembodiment a less aggressive therapy comprises delaying administrationof anti-HIV disease drugs and selecting and administering a singletherapy instead of a dual therapy. In one embodiment a less aggressivetherapy comprises selecting and administering a single therapy insteadof a dual therapy and selecting and administering less potent drugs. Inone embodiment a less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy and decreasingdosage of anti-HIV disease drugs. In one embodiment a less aggressivetherapy comprises selecting and administering a single therapy insteadof a dual therapy and decreasing the frequency of the dose schedule. Inone embodiment a less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy and shorteninglength of therapy. In one embodiment, less aggressive therapy comprisesselecting and administering a single therapy instead of a dual therapyand selecting and administering less potent drugs and decreasing drugdosage. In one embodiment, less aggressive therapy comprises selectingand administering a single therapy instead of a dual therapy andselecting and administering less potent drugs and decreasing doseschedule. In one embodiment, less aggressive therapy comprises selectingand administering a single therapy instead of a dual therapy, selectingand administering less potent drugs and shortening length of therapy. Inone embodiment, less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy, decreasingdrug dosage and decreasing dose schedule. In one embodiment, lessaggressive therapy comprises selecting and administering a singletherapy instead of a dual therapy, decreasing drug dosage and shorteninglength of therapy. In one embodiment, less aggressive therapy comprisesselecting and administering a single therapy instead of a dual therapy,decreasing dose schedule and shortening length of therapy. In oneembodiment, less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy, selecting andadministering less potent drugs, decreasing drug dosage, and decreasingdose schedule. In one embodiment, less aggressive therapy comprisesselecting and administering a single therapy instead of a dual therapy,selecting and administering less potent drugs, decreasing drug dosage,and shortening length of therapy. In one embodiment, less aggressivetherapy comprises selecting and administering a single therapy insteadof a dual therapy, selecting and administering less potent drugs,decreasing dose schedule, and shortening length of therapy. In oneembodiment, less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy, decreasingdrug dosage, decreasing dose schedule, and shortening length of therapy.In one embodiment, less aggressive therapy comprises selecting andadministering a single therapy instead of a dual therapy, selecting andadministering less potent drugs, decreasing drug dosage, decreasing doseschedule, and shortening length of therapy. In one embodiment a lessaggressive therapy comprises selecting and administering a singletherapy instead of a dual therapy.

In one embodiment a less aggressive therapy comprises selecting andadministering a dual therapy instead of a triple therapy. In oneembodiment a less aggressive therapy comprises selecting andadministering a dual therapy instead of a triple therapy and delayingadministration of anti-HIV disease drugs. In one embodiment a lessaggressive therapy comprises selecting and administering a dual therapyinstead of a triple therapy and selecting and administering less potentdrugs. In one embodiment a less aggressive therapy comprises selectingand administering a dual therapy instead of a triple therapy anddecreasing dosage of anti-HIV disease drugs. In one embodiment a lessaggressive therapy comprises selecting and administering a dual therapyinstead of a triple therapy and decreasing the frequency of the doseschedule. In one embodiment a less aggressive therapy comprisesselecting and administering a dual therapy instead of a triple therapyand shortening length of therapy. In one embodiment, less aggressivetherapy comprises selecting and administering a dual therapy instead ofa triple therapy, selecting and administering less potent drugs anddecreasing drug dosage. In one embodiment, less aggressive therapycomprises selecting and administering a dual therapy instead of a tripletherapy, selecting and administering less potent drugs and decreasingdose schedule. In one embodiment, less aggressive therapy comprisesselecting and administering a dual therapy instead of a triple therapy,selecting and administering less potent drugs and shortening length oftherapy. In one embodiment, less aggressive therapy comprises selectingand administering a dual therapy instead of a triple therapy, decreasingdrug dosage and decreasing dose schedule. In one embodiment, lessaggressive therapy comprises selecting and administering a dual therapyinstead of a triple therapy, decreasing drug dosage and shorteninglength of therapy. In one embodiment, less aggressive therapy comprisesselecting and administering a dual therapy instead of a triple therapy,decreasing dose schedule and shortening length of therapy. In oneembodiment, less aggressive therapy comprises selecting andadministering a dual therapy instead of a triple therapy, selecting andadministering less potent drugs, decreasing drug dosage, and decreasingdose schedule. In one embodiment, less aggressive therapy comprisesselecting and administering a dual therapy instead of a triple therapy,selecting and administering less potent drugs, decreasing drug dosage,and shortening length of therapy. In one embodiment, less aggressivetherapy comprises selecting and administering a dual therapy instead ofa triple therapy, selecting and administering less potent drugs,decreasing dose schedule, and shortening length of therapy. In oneembodiment, less aggressive therapy comprises selecting andadministering a dual therapy instead of a triple therapy, decreasingdrug dosage, decreasing dose schedule, and shortening length of therapy.In one embodiment, less aggressive therapy comprises selecting andadministering a dual therapy instead of a triple therapy, selecting andadministering less potent drugs, decreasing drug dosage, decreasing doseschedule, and shortening length of therapy.

In one aspect of the present application where there is an increasedlevel of level of sIP-10 or ratio of sIP-10 to total IP-10 or activityof DPPIV with respect to a reference level, treatment comprises a moreaggressive therapy than a reference therapy. In one embodiment a moreaggressive therapy comprises earlier administration of anti-HIV diseasedrugs. In one embodiment a more aggressive therapy comprises increaseddosage of anti-HIV disease drugs. In one embodiment a more aggressivetherapy comprises increased length of therapy. In one embodiment a moreaggressive therapy comprises increased frequency of the dose schedule.In one embodiment, more aggressive therapy comprises selecting andadministering more potent drugs and increasing drug dosage. In oneembodiment, more aggressive therapy comprises selecting andadministering more potent drugs and increasing dose schedule. In oneembodiment, more aggressive therapy comprises selecting andadministering more potent drugs and increasing length of therapy. In oneembodiment, more aggressive therapy comprises increasing drug dosage andincreasing dose schedule. In one embodiment, more aggressive therapycomprises increasing drug dosage and increasing length of therapy. Inone embodiment, more aggressive therapy comprises increasing doseschedule and increasing length of therapy. In one embodiment, moreaggressive therapy comprises selecting and administering more potentdrugs, increasing drug dosage, and increasing dose schedule. In oneembodiment, more aggressive therapy comprises selecting andadministering more potent drugs, increasing drug dosage, and increasinglength of therapy. In one embodiment, more aggressive therapy comprisesselecting and administering more potent drugs, increasing dose schedule,and increasing length of therapy. In one embodiment, more aggressivetherapy comprises increasing drug dosage, increasing dose schedule, andincreasing length of therapy. In one embodiment, more aggressive therapycomprises selecting and administering more potent drugs, increasing drugdosage, increasing dose schedule, and increasing length of therapy.

In one embodiment a less aggressive therapy comprises selecting andadministering a dual therapy instead of a single therapy. In oneembodiment a more aggressive therapy comprises selecting andadministering a dual therapy instead of a single therapy and earlieradministration of anti-HIV disease drugs. In one embodiment a moreaggressive therapy comprises selecting and administering a dual therapyinstead of a single therapy and increased dosage of anti-HIV diseasedrugs. In one embodiment a more aggressive therapy comprises selectingand administering a dual therapy instead of a single therapy andincreased length of therapy. In one embodiment a more aggressive therapycomprises selecting and administering a dual therapy instead of a singletherapy and increased frequency of the dose schedule. In one embodiment,more aggressive therapy comprises selecting and administering a dualtherapy instead of a single therapy, selecting and administering morepotent drugs and increasing drug dosage. In one embodiment, moreaggressive therapy comprises selecting and administering a dual therapyinstead of a single therapy, selecting and administering more potentdrugs and increasing dose schedule. In one embodiment, more aggressivetherapy comprises selecting and administering a dual therapy instead ofa single therapy, selecting and administering more potent drugs andincreasing length of therapy. In one embodiment, more aggressive therapycomprises selecting and administering a dual therapy instead of a singletherapy, increasing drug dosage and increasing dose schedule. In oneembodiment, more aggressive therapy comprises selecting andadministering a dual therapy instead of a single therapy, increasingdrug dosage and increasing length of therapy. In one embodiment, moreaggressive therapy comprises selecting and administering a dual therapyinstead of a single therapy, increasing dose schedule and increasinglength of therapy. In one embodiment, more aggressive therapy comprisesselecting and administering a dual therapy instead of a single therapy,selecting and administering more potent drugs, increasing drug dosage,and increasing dose schedule. In one embodiment, more aggressive therapycomprises selecting and administering a dual therapy instead of a singletherapy, selecting and administering more potent drugs, increasing drugdosage, and increasing length of therapy. In one embodiment, moreaggressive therapy comprises selecting and administering a dual therapyinstead of a single therapy, selecting and administering more potentdrugs, increasing dose schedule, and increasing length of therapy. Inone embodiment, more aggressive therapy comprises selecting andadministering a dual therapy instead of a single therapy, increasingdrug dosage, increasing dose schedule, and increasing length of therapy.In one embodiment, more aggressive therapy comprises selecting andadministering a dual therapy instead of a single therapy, selecting andadministering more potent drugs, increasing drug dosage, increasing doseschedule, and increasing length of therapy.

In one embodiment a more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy. In oneembodiment a more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy and earlieradministration of anti-HIV disease drugs. In one embodiment a moreaggressive therapy comprises selecting and administering a tripletherapy instead of a dual therapy and increased dosage of anti-HIVdisease drugs. In one embodiment a more aggressive therapy comprisesselecting and administering a triple therapy instead of a dual therapyand increased length of therapy. In one embodiment a more aggressivetherapy comprises selecting and administering a triple therapy insteadof a dual therapy and increased frequency of the dose schedule. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, selecting andadministering more potent drugs and increasing drug dosage. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, selecting andadministering more potent drugs and increasing dose schedule. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, selecting andadministering more potent drugs and increasing length of therapy. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, increasingdrug dosage and increasing dose schedule. In one embodiment, moreaggressive therapy comprises selecting and administering a tripletherapy instead of a dual therapy, increasing drug dosage and increasinglength of therapy. In one embodiment, more aggressive therapy comprisesselecting and administering a triple therapy instead of a dual therapy,increasing dose schedule and increasing length of therapy. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, selecting andadministering more potent drugs, increasing drug dosage, and increasingdose schedule. In one embodiment, more aggressive therapy comprisesselecting and administering a triple therapy instead of a dual therapy,selecting and administering more potent drugs, increasing drug dosage,and increasing length of therapy. In one embodiment, more aggressivetherapy comprises selecting and administering a triple therapy insteadof a dual therapy, selecting and administering more potent drugs,increasing dose schedule, and increasing length of therapy. In oneembodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, increasingdrug dosage, increasing dose schedule, and increasing length of therapy.In one embodiment, more aggressive therapy comprises selecting andadministering a triple therapy instead of a dual therapy, selecting andadministering more potent drugs, increasing drug dosage, increasing doseschedule, and increasing length of therapy.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description, and from the claims.

EXAMPLES

Total IP-10 Expression is Rapidly Controlled During Non-Pathogenic SIVInfection

We searched for factors involved in the lack of chronic immuneactivation in the AGM animal model. We showed that AGM, during acute SIVinfection, display signs of inflammation. For instance, AGM produceIFN-α and IFN-γ upon SIVagm infection. However this inflammation is onlyobserved during the first weeks post-infection and does not persist(Kornfeld et al., J Clin Invest., 115(4): 1082-1091, 2005; Ploquin etal., Retrovirology, 3: 37, 2006; Diop et al., J Virol., 82(11):5145-5152, 2008). Our data therefore revealed that AGM do not lack butrather regulate inflammation.

The capacity of natural hosts to mount an inflammation during acuteinfection was highly disputed in the literature. In order to furtherdemonstrate that AGM do not lack the capacity to sensor SIVagm infectionand mount an innate immune response, we performed a trancriptomeanalysis. Among other genes, we measured the expression profiles ofInterferon-stimulated genes (ISG), which are considered as surrogatemarkers for IFN activity. We and others demonstrated a rapid and stronginduction of ISGs, including IP-10 (Jacquelin et al., J Clin Invest,119(12): 3544-3555, 2009; Favre et al., PLoS Pathog., 5(2): e1000295,2009). Interestingly, in contrast to pathogenic SIVmac infection inmacaques, the expression of IP-10 was transient in AGM, in contrast tomacaques, where IP-10 expression remains highly elevated (Jacquelin etal., J Clin Invest, 119(12): 3544-3555, 2009; Favre et al., PLoSPathog., 5(2): e1000295, 2009) (FIG. 1).

Plasma Total IP-10 Levels During Acute HIV-1 Infection are StrongerPredictors of Disease Progression than Viremia and CD4⁺ T Cell Counts

The data on the differences between pathogenic and non-pathogenic SIVinfection has lead us raise the hypothesis that uncontrolledinflammation and IFN response at the end of acute HIV infection could beof bad prognosis for disease progression in HIV-infected individuals.The concentrations of 28 plasma proteins, including IFN-I inducibleproteins and anti-inflammatory cytokines, were quantified during acuteHIV-1 infection of 133 non-treated patients. This was a retrospectivestudy, and the disease progression profiles of each patient were known(Liovat et al., Keystone Symposium on HIV Evolution, Genomics andPathogenesis. Whistler, Canada; 2011; Liovat et al., 6th IAS Conferenceon HIV pathogenesis, treatment and prevention. Rome; 2011; Liovat etal., PLoS One, 7(10): e46143, 2012). This study revealed for the firsttime an association between inflammation in acute infection and T cellactivation at set-point (i.e. 6 months post-infection). Moreover, IP-10was an independent predictor of rapid disease progression. Of note, whenmeasured in acute HIV-1 infection, IP-10 robustness of prediction wasstronger than that of viremia or CD4⁺ T cell counts.

Only two other studies had analysed before the predictive capacity ofinflammatory molecules during acute HIV infection to predict subsequentdisease progression. Roberts et al did not identify an association ofIP-10 with disease progression (Roberts et al., Aids, 24(6): 819-31,2010), while Jiao showed a predictive value of early IP-10 levels forCD4⁺ T cell loss in a cohort of Chinese patients (Jiao et al., ViralImmunol., 25(4): 333-337, 2012). Other studies had evaluated IP-10 inchronic phase of infection or after treatment and had a found a negativecorrelation with CD4⁺ T cell counts and a strong association withviremia levels (Kamat et al., PLoS One, 7(2): e30881, 2012; Keating etal., Aids, 25(15): 1823-32, 2011). Our data indicate for the first timethat when measured in acute HIV-1 infection, IP-10 robustness ofprediction is stronger than that of VL and CD4⁺ T cell counts (Liovat etal., PLoS One, 7(10): e46143, 2012) (FIG. 2).

The Plasma IP-10 Level During Acute HIV-1 Infection is a StrongerPredictor of Disease Progression than the Viral Reservoir Size

In order to further demonstrate the robustness of prediction of IP-10,we wanted to compare it to other markers that have been associated withdisease progression. We already compared it above to viral RNA levels inplasma. In recent times, numerous studies stressed the importance of theviral DNA levels, which are associated with the number of infectedcells. Indeed, the viral DNA copy numbers correspond to the so-calledviral reservoir. The size of this viral reservoir reflects the host'scapacity to control viral replication, infection and spread. The size ofthe viral reservoir is remarkably low in HIV controllers and inpost-treatment controllers. It has been suggested that anearly/immediate start of HAART after HIV infection is of benefit for thepatients because it might limit the viral reservoir. A study carried outon 22 patients enrolled during acute HIV infection, showed that afterseveral months of HAART, among 22 analytes measured, only IP-10 differedsignificantly between treated and non-treated participants (Gay et al.,PLoS One, 6(5): e19617, 2011).

We are currently collaborating with Pr. Christine Rouzioux (HôpitalNecker). Her team had quantified the viral DNA load in the samepatients, where we had quantified total IP-10 levels. The levels ofIP-10 and viral DNA in acute infection were correlated (R=0.35p=0.0001). In multivariate analyses, the plasma levels of IP-10 duringacute infection remained highly predictive of rapid progression towardsAIDS (p=0.003), while viral DNA levels were not (p=0.47). This supportsour previous findings on IP-10 being a robust and early predictor ofdisease progression (FIG. 2).

The Plasma IP-10 Level Before HIV-1 Infection Predicts the Rate of CD4+T Cell Loss after Infection

In order to validate our results in an independent cohort, we contactedthe coordinator of the Amsterdam Cohort studies (ACS) in Netherlands.The ACS are recognized as one of the oldest and biggest HIV-1 cohorts. Arare feature is that for hundreds of HIV patients, blood samplescollected before infection are available in the ACS. We thus quantifiedIP-10 in plasma at different time points before and after HIV-1infection in 136 non-treated patients. The IP-10 levels before infectioncorrelated with the levels 6 months after HIV infection (R=0.37p=0.003). Of note, the levels of IP-10 within the 25 months beforeinfection were predictive of rapid disease progression upon HIV-1infection (OR=3.18 95%1.06-9.55 p=0.03).

The Ratio of the Antagonist Form of IP-10 (Short IP-10) is Decreased inAcute HIV-1 Infection in Patients Who Subsequently Progress RapidlyTowards AIDS

By measuring total IP-10, as we did above, one cannot distinguishbetween distinct IP-10 forms, i.e. short and long forms. During HCVinfection, high levels of plasma IP-10 are predictive of the failure torespond to peg-IFN-a₂/RBV therapy (Casrouge et al., J Clin Invest.,121(1): 308-317, 2011). Investigators in the Albert laboratorydemonstrated that these elevated IP-10 levels are mainly in the shortantagonist form. This increased IP-10 antagonism prevents trafficking ofCXCR3+ lymphocytes capable to control HCV replication, to the liver,thus diminishing HCV control and explains the failure of response topeg-IFN-a₂/RBV treatment ((Casrouge et al., J Clin Invest., 121(1):308-317, 2011; Casrouge et al., Clin Exp Immunol., 167(1): 137-148,2012). We hypothesized that progressive HIV/SIV infections areassociated with decreased IP-10 antagonism, thus enhancing CXCR3+lymphocyte trafficking into lymphoid organs and the gut mucosaamplifying inflammation during HIV/SIV infection. This model would bethe opposite picture of what has been suggested for HCV infection, wherehigh levels of IP-10 antagonist form are harmful, while in HIV infectionthey would limit inflammation and play a protective role.

To address this question we started to evaluate the concentrations ofIP-10 antagonist form in plasma from acutely HIV-infected individuals(ANRS Primo C06 cohort). Using a standardized ELISA developed in MatthewAlbert's laboratory consisting of a specific antibody raised against theIP-10 antagonist form (Casrouge et al., J Clin Invest., 121(1): 308-317,2011), we performed a preliminary evaluation of 53 samples. The ratio ofshort versus total IP-10 antagonist was decreased during acute HIV-1infection in rapid progressors compared to the other HIV-infectedpatients, and also compared to healthy donors (p<0.01) (FIG. 3).

There was a trend for a positive correlation (R=0.31 p=0.0575) betweenshort IP-10 in acute infection and the CD4 T-cell counts at set-point 6months later (M6). Moreover, the ratios of short against total IP-10 inacute infection negatively correlated (R=0.4764 p=0.0494) with CD8T-cell activation at M6.

The Activity of DPPIV is Reduced During Acute Infection of Rapid DiseaseProgressors

Next, we wondered which factors could be responsible for the distinctlevels of IP-10 among the individuals. The Dipeptidyl peptidase IV(DPPIV) is known to cleave IP-10 into the short IP-10 form. It hasalready been shown that DPPIV is decreased in the blood of patientschronically infected by HIV (Blazquez et al., J Immunol., 149(9):3073-3077, 1992; De Pasquale et al., Acta Haematol., 81(1): 19-21, 1989;Gougeon et al., Res Immunol., 147(1): 5-8, 1996; Vanham et al., J AcquirImmune Defic Syndr, 6(7): 749-757, 1993). We quantified DPPIV for thefirst time in acute HIV-1 infection. Importantly, we evaluated DPPIV inpatients showing three distinct types of disease progression profile:slow progression, normal progression and rapid progression towards AIDS.Soluble DPPIV activity was measured in 53 patients of the ANRS PrimoCohort No 6 and 134 patients of the ACS. The plasma levels of solubleDPPIV were significantly reduced during acute HIV infection in bothcohorts (FIGS. 3 and 4). The levels were significantly lower in rapiddisease progressors than in normal or slow progressors. Indeed thelevels measured in acute HIV-1 infection were predictive of rapiddisease progression (OR=3.27 95%1.05-10.16 p=0.04). Further, the plasmaslevels of soluble DPPIV (measured in sera samples from the ACS cohort)at 6 months after seroconversion had predictive value for rapid diseaseprogression (OR=1.88 95%1.03-3.45 p=0.04), The robustness was weaker asobserved in the ANRS cohort might be due to measurements carried out ata later time point. Thus, our study reveals distinct levels of DPPIVaccording to the disease progression profile.

In line with this, the plasma levels of soluble DPPIV (measured inplasma samples from the ANRS cohort) at primary HIV infection correlatednegatively with viremia as well as with T cell activation at set-point((p=0.003, R=−0.76 and p=0.049, R=−0.42, respectively).

Ultimately, these data suggest that decreased levels of IP-10 antagonistform and sDPPIV activity badly influence the viral and immunologicset-points. In other words, preserving sDPPIV activity leading to highlevels of IP-10 antagonist would be of good prognosis for HIV-1infection.

Higher Inflammation in the Gut of SIV-Infected Macaques is Associatedwith Lower Expression of DPPIV

In order to confirm the link between DPPIV and AIDS pathogenesis, weinitiated a study to address this in simian models. We sampled guttissue from SIV-infected MAC (n=6) and AGM (n=5) at day 65 p.i. Thiscorresponds to an interesting time point as it is situated shortly afteracute infection, when IP-10 levels are already differently regulatedbetween MAC and AGM (Jacquelin et al., J Clin Invest, 119(12):3544-3555, 2009). We harvested fragments of jejunum, ileum, colon andrectum. We quantified IP-10 gene expression levels in enrichedintra-epithelial (IEC) and CD4⁺ cells of each of these compartments.IP-10 was significantly more expressed in the high intestine than incolon/rectum. We also detected an over-representation of CXCR3 genetranscripts in CD4+ cells from the MAC high intestine.

Equally in accordance with our hypothesis, DPP4 gene expression was lesspronounced in CD4⁺ cells from MAC than in AGM (FIG. 5). There was asignificant negative correlation between DPPIV in CD4⁺ and in IEC withIP-10 gene expression in the CD4⁺ compartment of the high intestine(FIG. 5).

Altogether, lower IP-10 inflammation in the gut was associated withhigher levels of DPPIV.

DPPIV Levels in the Jejunum Correlated with Those in Plasma During SIVInfection

The intestine corresponds to the major site of replication for HIV andSIV. According to the literature, virus-host interactions in theintestine have a major impact on the outcome of infection (Douek, TopHIV Med., 15(4): 114-117, 2007). With the help of the animal model, weaddressed the question whether the levels of IP-10 and DPPIV in theblood are representative of the respective expression levels in the gutduring infection. We quantified IP-10 and soluble DPPIV activity in theplasma of the same animals for which we had analyzed the gut tissues.The activity of soluble DPPIV in the plasma of rapidly progressingmacaques seemed lower than in AGM. IP-10 and DPPIV levels in the jejunumcorrelated with their respective levels in plasma (R=0.77, p=0.03;R=0.79, p=0.009, respectively) (FIG. 6). This is in support for IP-10and DPPIV in blood as valuable markers reflecting mucosal inflammationin addition to their predictive values for rapid disease progression.

CONCLUSION

Altogether, this is the first time that DPPIV has been measured duringacute HIV-1 infection. Our studies in the animal model confirm the linkbetween DPPIV and disease progression. They indicate furthermore thatthe levels in the blood reflect ongoing pathogenic processes in thetissues.

This is also the first time that the short form of IP-10 has beenquantified during HIV infection. In line with the reduced levels ofDPPIV, those patients with the worst clinical prognosis, displayed thelowest ratios of short IP-10 as compared to the other HIV-infectedindividuals.

We claim:
 1. A method for selecting an anti-HIV therapy for a subjectidentified as being infected with HIV, said method comprising: a)measuring the sIP-10 level in a sample of the said subject by ELISA withan antibody recognizing sIP-10; b) measuring the total IP-10 level insaid sample of the said subject by ELISA with an antibody recognizinatotal IP-10; c) calculating the ratio of sIP-10 to total IP-10; d)determining that said subject has a high likelihood to develop AIDS ifthe ratio of step c) is less than the ratio of sIP-10 to total IP-10 ina healthy subject; e) selecting the therapy based on said likelihood,wherein the therapy is selected from the group consisting of: highlyactive antiretroviral therapy (HAART), protease inhibitors, fusioninhibitors, integrase inhibitors, co-receptor specific agents, 3TC, AZT,nevirapine, non-nucleoside analogue reverse transcriptase inhibitors,and nucleoside analogue reverse transcriptase inhibitors; and f)administering said therapy.
 2. The method of claim 1, further comprisinga prior step of obtaining said sample.
 3. The method of claim 1, whereinsaid sample is selected from the group of blood, plasma, lymph, bonemarrow fluid, pleural fluid, peritoneal fluid, spinal fluid, abdominalfluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites,urine, saliva, bronchial lavage, bile, sweat, tear, earflow, sputum,semen, vaginal flow, milk, amniotic fluid, or secretions of respiratory,intestinal or genitourinary tract.
 4. The method of claim 1, wherein thesubject is selected in the group of subjects havina T cell counts ofless than 200 cells/μL, comprised between 200 and 500 cells/μL, andabove 500 cells/μL.
 5. A method for selecting an anti-WV therapy for asubject identified as being infected with HIV, said method comprising:a) measuring the sIP-10 level in a sample of the said subject by ELBAwith an antibody recognizing sIP- 10; h) measuring the total IP-10 levelin said sample of the said subject by ELISA with an antibody recognizingtotal IP-10; c) calculating the ratio of sIP-10 to total IP-10; d)determining that the prognosis of said subject is had if the ratio ofstep c) is less than the ratio of sIP-10 to total IP-10 in a healthysubject; and e) selecting the therapy based on said prognosis, whereinwherein the therapy is selected from the group consisting of: highlyactive antiretroviral therapy (HAART), protease inhibitors, fusioninhibitors, integrase inhibitors, co-receptor specific agents, 3TC, AZT,nevirapine, non-nucleoside analogue reverse transcriptase inhibitors,and nucleoside analogue reverse transcriptase inhibitors; and f)administering said therapy.
 6. The method of claim 5, further comprisinga prior step of obtaining said sample.
 7. The method of claim 5, whereinsaid sample is selected from the group of blood, plasma, lymph, bonemarrow fluid, pleural fluid, peritoneal fluid, spinal fluid, abdominalfluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites,urine, saliva, bronchial lavage, bile, sweat, tears, ear flow, sputum,semen, vaginal flow, milk, amniotic fluid, or secretions of respiratory,intestinal or genitourinary tract.
 8. The method of claim 5, wherein thesubject is selected in the group of subjects having T cell counts ofless than 200 cells/μL, comprised between 200 and 500 cells/μL, andabove 500 cells/μL.